Development of a high-throughput RT-PCR based viral infectivity assay for monitoring the stability of a replicating recombinant Lymphocytic Choriomeningitis viral vector

•Development of a RT-qPCR-based infectivity assay for a live, recombinant replicating lymphocytic choriomeningitis virus (LCMV) viral vector.•A high positive correlation between the RT-qPCR and the “gold-standard” infectivity assays.•The infectivity RT-qPCR assay demonstrated increased sample throughput and improved assay flexibility.•The RT-qPCR assay can potentially be utilized as an in-process monitoring tool during LCMV vector manufacturing. Traditional virus infectivity titration methods for lymphocytic choriomeningitis virus (LCMV) are laborious, time-consuming, and low-throughput (e.g., focus forming unit (FFA) assay). In this report, we developed a high-throughput reverse transcription quantitative PCR (RT-qPCR)-based virus infectivity assay for relative quantitation of a live, recombinant replicating LCMV -based viral vector (TT1). This in vitro infectivity assay demonstrated a 4-log linear range for TT1 titer quantitation. A high positive Pearson correlation coefficient value (≥ 0.80) was obtained between the RT-qPCR vs. the “gold-standard” FFU assay when comparing the stability profiles of stressed TT1 vector samples. In addition to the RT-qPCR infectivity assay, the stability of the TT1 vector upon freeze-thaw stress was investigated further with complementary viral particle characterization techniques (e.g., TEM, NTA, MFI). Correlations between viral infectivity and particle measurements during forced degradation studies were observed to be specific to the TT1 vector and its various formulations and such results facilitated the rank-ordering of formulation conditions. Overall, this infectivity RT-qPCR method showed increased sample throughput and improved assay flexibility compared to traditional viral infectivity assays. These results are discussed in the context of enabling future TT1 vector formulation development work, and potential utilization as an in-process monitoring tool during TT1 vector manufacturing.